C4d – The Birmingham UK Experience

Desley Neil, Majid Mukadam, David Briggs*UHBNHSFT, NHSBT*

Birmingham

Method

• C4d staining performed routinely on all EMBs from May 2004.• May 2004 – May 2008

– 1443 biopsies in 163 patients with 166 transplants– reports for Grade of C4d staining

• 71 (4.9%) unreported – 14 missing , rest graded• Presence or absence DSA (luminex bead) – not routine• C3d staining on strong C4d cases• Pattern of inflammation reassessed in

• Strong C4d bx V highest C4d neg/weak• Busy, no, focal or diffuse infiltrate, Cells in / around capillary

Biopsy protocol• Protocol

– Weekly x 6– Fortnightly x 2– Monthly x 2– 6 weekly x 2 1year– 6 monthly x2 2 years– Yearly x1 3 years

• Indication– Symptomatic or change in echo

• Change of medication– 2-3 biopsy during transition and once established

C4d (& C3d) grading system

• 0 Negative• 1 Weak patchy staining• 2 Moderate staining• 3 Diffuse strong staining

(Looks like CD31 at low power)

• Immunoperoxidase Polyclonal Ab Biomedica

C4d

C3d

Demographics

49 (14-65)49 (14-63) 44 (19-65) Age

132 31 163

Biopsies

• Timing of biopsies– 171 days (0-5806 days) post-tx

• Number of biopsies / patient– 4 (1-40) biopsies

C4d staining

72%

15%

60%

Highest C4d grade / patient

12.3%

35.6%

22.1%29.5%

Death related to highest C4d6.3% 11.1% 20.7% 35%

Kruskal Wallis p<0.02

20.0015.0010.005.000.00

followup

1.0

0.8

0.6

0.4

0.2

0.0

Cu

m S

urv

ival

3-censored

2-censored

1-censored

0-censored

3

2

1

0highestC4d

Survival Functions

Follow up time v highest C4d grade

Follow up 0 1 2 3 missing

N= 48 36 58 20 1

Median(range) 8 yrs

(0.4-16.8)4.4 yrs(0.5-15.5)

3.6yrs(0.3-14.8)

6 yrs(0.6-15.6)

12.4yr

Kruskal Wallis p=0

Strong C4d

• 20 patients• 7 had DSA found• 6/7 (85.7%) with DSA died• 1 with lot of consecutive strong C4d

– serum not sent till after Plasma exchange = negative

– Retransplanted

• Others only HLA Ab tested inconsistently

DSA

• 19 (11.7%) patients• Found 7.1 (0-12.9) years post-tx• 13 (68.6%) class II and 6 (31.4%) class I and II

Demographics of DSA +/-

Age: DSA + 46 (14-59) DSA – 49 (17-65)

NS

Symptoms

• 5 (26.3%) asymptomatic• 14 (73.3%) symptomatic

– 3 IHD/graft vasculopathy– 11 syncope, heart failure

• 10 evidence of graft vasculopathy• 8 no evidence of graft vasculopathy• 1 don’t know

Death related to DSA47.4%18.8%10.5%

Death v DSA Wilcoxin p=0.000

Follow up time v DSA

Not tested Neg PosN= 19 125 19

Median(range) 6.1 yrs

(0.4-15.7)5.2 yrs(0.3-15.8)

8.6yrs(0.9-13.5)

Kruskal Wallis NS

Death8.8 (3-12.9) yrs post tx215 (7-1188) days post DSA found

DSA v highest C4d grade

Kruskal Wallis p=0

35%17.2%2.8%2.1%

DSA v Strong C4d +C3d grade

DSA /Patient with strong C4d +/- mod/strong C3d

60%

P<0.05

DSA in relation to C4d persistence

• Persistent strong 4/6 (66.7%)• Intermittent mod/strong 4/17 (23.5%)• Single strong (others neg/weak) 4/18 (22.2%)• Single bx = strong (no others bx) 3/7 (42.9%)• Nothing much 3/111 (2.7%)

Kruskal-Wallis p<0.02

C3d staining in relation to persistence of C4d

N=357 neg/weakN=42 strong

BUSY “B” PATTERN

Summary• C4d staining relatively uncommon• Both DSA and highest grade of C4d correlates with

death• Correlation between C4d and DSA –? improved by

C3d• Neither C4d or DSA in isolation is sensitive at a

single time point – C4d - comes and goes, precede inflam and symptoms– Repeat DSAs (- to + in 4/7)

• Using ISHLT criteria will miss 2/3 C4d positive cases

• same also if C3d mod/strong• Busy “B” with cells in or around vessels• C4d/C3d needs to be routine

C4d grade